Use of pdgfr-alpha as diagnostic marker for papillary thyroid cancer

ABSTRACT

Provided herein are methods for identifying a subject with an increased likelihood of developing or having metastatic papillary thyroid cancer (PTC), or a subject with an increased likelihood of developing or having recurrent PTC, and the treatment of such a subject.

RELATED APPLICATIONS

This application claims priority to U.S. Application No. 61/593,414filed Feb. 1, 2012, the contents all of which are hereby incorporated byreference in their entirety.

FIELD OF THE INVENTION

The field of the invention generally relates to compositions and methodsfor identifying a subject with an increased likelihood of developing orhaving metastatic papillary thyroid cancer (PTC), as well as thetreatment of such a subject population.

BACKGROUND OF THE INVENTION

Clinical or radiographic identification of thyroid nodules requiresassessment for malignancy through tissue biopsy.^(1,2) Typicallyobtained through fine needle aspiration (FNA), thyroid nodule biopsy candistinguish cancer from benign disease in approximately 65% of cases inlarge series and is considered essential in the workup of any thyroidnodule.^(1,3) Improvements in the accuracy of tissue biopsy haveutilized ultrasound and a standardized pathology reporting system.⁴

Efforts to extend the utility of this tissue resource are now focusedprimarily on molecular methods to better predict the natural history ofdisease and tailor patient therapy.⁵ Papillary thyroid cancer (PTC),comprising more than 80% of all thyroid cancer cases, has a highpropensity for spread within the lymphatic system.⁶ However, oneimportant limitation of current FNA assessment is that no information isprovided on the metastatic potential of thyroid malignancy.^(3,5,7) Upto 30% of papillary thyroid carcinoma cases demonstrate lymphaticmetastases which, if untreated through surgery or radioactive iodineablation, may lead to recurrent disease in the central or lateralneck.^(6,8-10) Patients with metastatic or recurrent PTC often requiremultiple surgical resections and radioactive iodine ablative treatmentswith associated increased morbidity.⁹⁻¹² Predicting aggressive ormetastatic variants through tissue biopsy could direct surgeons toprophylactic neck dissections and guide adjuvant radioiodine therapy todecrease the risk of local and regional recurrence and improvequality-of-life.¹¹⁻¹³

In the drive to develop molecular techniques to make more personalizedchoices for patient diagnosis and therapy, a large number of studies onthe mitogen-activated protein kinase (MAPK/ERK) signaling pathway havebeen undertaken to understand the pathogenesis of thyroidcancer.^(14,15) It is understood that rearrangements of tyrosine kinasegenes RET/PTC and activating mutations of the BRAF or RAS commonlyactivate the MAPK/ERK pathway.¹⁶⁻¹⁸ BRAF, an isoform of a class ofserine-threonine kinases, is also a potent activator of this pathway andthe V600E mutation is an important and well conserved mutation inpapillary thyroid cancer.¹⁷ Activating mutations of the RAS genes,namely H-/K-/N-ras, also play an important role in the pathogenesis ofpapillary thyroid cancer through the MAPK/ERK pathway.^(18,19) Othergenotype-phenotype correlations have been undertaken in thyroid cancerusing gene arrays to develop predictive tools based on galectin-3, cellcycle proteins and apoptotic markers.¹⁹⁻²⁵ RET/PTC translocations andactivating mutations of BRAF and RAS genes are considered clinicallyrelevant markers that have been endorsed for use by the American ThyroidAssociation in the diagnosis of thyroid cancer when tumor cytology isindeterminate.^(5,25) At this time these genetic testing regimes areutilized selectively in only a few high-volume centers. While studies ofdiagnostic biomarkers for thyroid cancer dominate the literature,relatively few studies have examined the pathways and processesmediating lymphatic or distant spread in thyroid cancer. Research intobiomarkers for lymphatic metastases reveal a number of changes in cellcycle proteins (cyclin D1), angiogenesis (vascular endothelial growthfactor-VEGF), and metalloproteinases (MMP-2) but none are clinicallyaccepted for use.²⁶⁻²⁹

Platelet derived growth factors (PDGFs) are a family of peptides thatbind to tyrosine kinase receptors (PDGF subunits α and β) and stimulatecell survival, growth, and proliferation.³⁰ PDGF promotes the epithelialto mesenchymal transition (EMT), an important process in tumormetastases, and complements the function of VEGF inangiogenesis.^(30,31)

It is, therefore, desirable to provide compositions and/methods foridentifying a subject with an increased likelihood of developing orhaving metastatic papillary thyroid cancer (PTC).

This background information is provided for the purpose of making knowninformation believed by the applicant to be of possible relevance to thepresent invention. No admission is necessarily intended, nor should itbe construed, that any of the preceding information constitutes priorart against the present invention.

SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention there is providedherein a method for identifying a subject with an increased likelihoodof developing or having metastatic papillary thyroid cancer (PTC), or asubject with an increased likelihood of developing or having recurrentPTC.

In accordance with one aspect of the present invention, there isprovided a method for treating a subject with or suspected of havingpapillary thyroid cancer, comprising: a) obtaining a tumor sample from asubject having thyroid cancer; b) processing said sample; c) performingan analyte binding assay configured to detect a biomarker in saidprocessed tumor sample by introducing the processed tumor sample into anassay instrument which (i) contacts a reagent which specifically bindsfor detection of the biomarker within the tumor sample, and (ii)generates one or more assay results indicative of binding of saidbiomarker, d) administering a treatment for papillary thyroid cancer tosaid subject when the amount of the biomarker in the sample is greaterthan that in a control sample, wherein said biomarker is PDGFR-α.

In accordance with one aspect of the present invention, there isprovided a method comprising: a) obtaining a sample from a subject withthyroid cancer; b) processing said sample; c) performing an analytebinding assay comprising contacting the processed sample with a reagentto form a complex between the reagent and the biomarker present in thesample; d) generating a result using instrumentation configured todetect said complex, said result indicative of the amount orconcentration of said complex formed to determine the amount orconcentration of said biomarker in the sample; and e) administering atreatment for papillary thyroid cancer to said subject when the amountof the biomarker in the sample is greater than that in a control sample,wherein said biomarker is PDGFR-α.

In accordance with one aspect of the present invention, there isprovided a method comprising: a) performing an analyte binding assaycomprising contacting a processed sample, said processed sample obtainedfrom a subject with thyroid cancer, with a reagent to form a complexbetween the reagent and the biomarker present in the sample; b)generating a result indicative of the amount or concentration of saidcomplex formed to determine the amount or concentration of saidbiomarker in the sample; and c) administering a treatment for papillarythyroid cancer to said subject when the amount of the biomarker in thesample is greater than that in a control sample, wherein said biomarkeris PDGFR-α.

In some examples, said biomarker is a biomarker protein, a biomarkertranscript, or biomarker activity.

In some examples, said analyte binding assay in an immunoassay.

In some examples, wherein said immunoassay is immunohistochemistry.

In some examples, said analyte binding assay is an RNA detecting assay.

In some examples, said RNA detecting assay comprises RT-PCR or in situhybridization.

In some examples, wherein said immunohistochemistry is performed with anautomated system, or a manual system.

In some examples, said assay results quantitative or semi-quantitative.

In some examples, wherein said processing comprises formalin fixing,paraffin-embedding, snap freezing, treated to isolate DNA, RNA, orprotein, or any combination thereof, said sample.

In some examples, said treatment comprises surgical resection, radiotherapy, chemotherapy, or combinations thereof.

In some examples, said radio therapy comprises radio iodine ablativetherapy.

In some examples, said chemotherapy comprises a tyrosine kinaseinhibitor such as sorafenib, sunitinib, axitimb, or motisanib.

In some examples, said treatment comprises administering an inhibitor ofPDGFR-α to said subject.

In some examples, said inhibitor comprises, an RNA interferencemolecule, a small molecule, nucleic acid, an antibody, a peptide, apharmaceutical composition, an aptamers, or combinations thereof.

In some examples, said RNA interference molecule comprises a RNAimolecule, a siRNA molecule, or a shRNA molecule.

In accordance with one aspect of the present invention, there isprovided a system for treating a subject with or suspected of havingpapillary thyroid cancer, comprising: a) a reagent which specificallybinds for detection of PDGFR-α in a tumor sample from a patient withthyroid cancer, and b) an assay instrument configured to receive a tumorsample and contact the reagent with the tumor sample, and to generateone or more assay result indicative of binding said reagent with thePDGFR-α within the tumor sample which is assayed for specific binding.

In some examples, said assay instrument comprises a detector set todetect a complex between said reagent and the PDGFR-α within the tumoursample, and wherein the instrument generates an assay results.

In some examples, the reagent is specific for PDGFR-α protein, PDGFR-αtranscript, or PDGFR-α activity.

In some examples, the system further comprising a treatment forpapillary thyroid cancer for said subject when the amount of the PDGFR-αin the sample is greater than that in a control sample.

In some examples, said treatment comprises surgical resection, radiotherapy, chemotherapy, or combinations thereof.

In some examples, said radio therapy comprises radio iodine ablativetherapy.

In some examples, said chemotherapy comprises, sorafenib, sunitinib,axitimb, or motisanib.

In some examples, said treatment comprises an inhibitor of PDGFR-α tosaid subject.

In some examples, said inhibitor comprises, an RNA interferencemolecule, a small molecule, nucleic acid, an antibody, a peptide, apharmaceutical composition, an aptamers, or combinations thereof.

In some examples, said RNA interference molecule comprises a RNAimolecule, a siRNA molecule, or a shRNA molecule.

In accordance with one aspect of the present invention, there isprovided a kit for treating a subject with or suspected of havingpapillary thyroid cancer, comprising: a) a reagent for performing ananalyte binding assay comprising contacting a processed sample from asubject with thyroid cancer with said reagent to form a complex betweenthe reagent and a biomarker present in the sample, wherein saidbiomarker is PDGFRα; and b) instructions for treating a subject with orsuspected of having papillary thyroid cancer according to the methods asdescribed herein.

In some examples, said reagent comprises an agent which binds to PDGFRαtranscript or PDGFRα protein.

In some examples, said reagent comprises an antibody.

In accordance with one aspect of the present invention, there isprovided use of an inhibitor of PDGFRα for the treatment of a subjectwith or suspected of having metastatic or recurrent PTC.

In some examples, wherein said subject is determined as having orsuspected as having metastatic or recurrent PTC by performing a) ananalyte binding assay comprising contacting a processed sample from saidsubject with a reagent to form a complex between the reagent and abiomarker present in the sample; and b) generating a result usinginstrumentation configured to detect said complex, said resultindicative of the amount or concentration of said complex formed todetermine the amount or concentration of said biomarker in the sample;wherein said subject is determined as having or suspect as having PCTwhen the amount of the biomarker in said processed sample is greaterthan a control.

In some examples, said biomarker is a biomarker protein, a biomarkertranscript, or biomarker activity.

In some examples, said analyte binding assay in an immunoassays.

In some examples, said immunoassay is immunohistochemistry.

In some examples, said analyte binding assay is an RNA detecting assay.

In some examples, said RNA detecting assay comprises RT-PCR or in situhybridization.

In some examples, said immunohistochemistry is performed with anautomated system, or a manual system.

In some examples, said assay results quantitative or semi-quantitative.

In some examples, said processing comprises formalin fixing, orparaffin-embedding, or both formalin fixing and paraffin-embedding, saidsample.

In some examples, said inhibitor comprises, an RNA interferencemolecule, a small molecule, nucleic acid, an antibody, a peptide, apharmaceutical composition, an aptamers, or combinations thereof.

In some examples, said RNA interference molecule comprises a RNAimolecule, a siRNA molecule, or a shRNA molecule.

In some examples, said use further comprising the use of radio therapy,chemotherapy, or combinations thereof.

In some examples, said radio therapy comprises radio iodine ablativetherapy.

In some examples, wherein said chemotherapy comprises a tyrosine kinaseinhibitor.

In some examples, wherein said chemotherapy comprises sunitinib,axitimb, or motisanib.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention will now be described, by way ofexample only, with reference to the attached Figures, wherein:

FIG. 1 shows representative immunohistochemical stains of PDGFR-α inpapillary thyroid cancer (PTC) primary tumors without nodal metastasesat low power (A) (100×) and high power (B) (400×), primary tumors withnodal metastases are shown (C) (100×) and (D) (400×);

FIG. 2 shows Western blots of PDGFR-α and -β in patient primary tumorslacking nodal metastases (#1-7, far left) compared to those with nodalmetastases (#8-14, middle section), corresponding metastatic diseasedeposits from patients #8-14 are shown in the far right section;

FIG. 3 shows Western blots of primary cell culture obtained from a lymphnode specimen with metastatic papillary thyroid cancer confirmed byhistology;

FIG. 4 shows Western blots documenting the activation status of theMAPK/ERK and PI3K/Akt pathways in primary tumours lacking nodalmetastases (#1-7, far left) and those with nodal metastases (#8-14,middle section), and corresponding metastatic disease deposits frompatients #8-14 are shown in the far right section;

FIG. 5 shows Western blots of PDGFR configuration in the papillarythyroid cancer cell lines;

FIG. 6 shows Cytoselect assay results (in triplicate) for invasivepotential of TPC-1 (A), 8305C (B), and BCPAP (C) cell lines;

FIG. 7 shows Cytoselect invasion assays with or without PDGFR-α siRNAfor TPC-1 cell line (A) with corresponding cell viability assessment(B), invasion assays for 8305C cell line with PDGFR-α siRNA shown in (C)with corresponding cell viability experiment (D);

FIG. 8 show the corresponding Western blots of the TPC-1 (A) and 8305C(B) cell lines for the invasion assay as shown in FIG. 6;

FIG. 9 shows disrupted invasive potential of TPC-1 cells with smallmolecule inhibition of (A) PI3K/Akt (Ly294002) or (B) MAPK/ERK (U0126)pathways, and bar graphs in (C) and (D);

FIG. 10 depicts the selective knock down of the PDGFR-alpha and -betasubunits in the TPC-1 cell lines;

FIG. 11 shows that knockdown of the PDGFR-beta subunit increases colonyformation in the TPC-1 cell line while selective PDGF-alpha expressionincreases cell size in the TPC-1 and BCPAP cell lines;

FIG. 12 shows that PDGFR-alpha subunit drives cell migration in TPC-1,BCPAP and 8305C cell lines.

FIG. 13 shows knock down of PDGFR-alpha or -beta subunits results inopposing effects in tumour formation on a mice xenograft model;

FIG. 14 shows immunohistochemical and immunoblot results from mousexenographts; and

FIG. 15 shows that PDGFR-alpha mRNA levels are greater in metastaticspecimens than in primary tumours.

In the Detailed Description that follows, the numbers in bold face typeserve to identify the component parts that are described and referred toin relation to the drawings depicting various embodiments of theinvention. It should be noted that in describing various embodiments ofthe present invention, the same reference numerals have been used toidentify the same of similar elements. Moreover, for the sake ofsimplicity, parts have been omitted from some figures of the drawings.

DETAILED DESCRIPTION

As will be described in more detail below, in one embodiment, there isprovided herein a method for identifying a subject with an increasedlikelihood of developing or having metastatic cancer, or a subject withan increased likelihood of developing or having recurrent cancer. Thereis also provided the treatment of such a subject population.

In one embodiment, there is provided herein a method for identifying asubject with an increased likelihood of developing or having metastaticpapillary thyroid cancer (PTC), or a subject with an increasedlikelihood of developing or having recurrent PTC. There is also providedmethods of treatment of this subject population.

In some examples, the subject is at risk for PTC, or is suspected ofhaving PTC, and/or has been diagnosed with PTC.

The term “subject at risk for PTC” as used herein, refers to a subjectwith one or more risk factors for developing PTC. Risk factors include,but are not limited to, gender, age, genetic predisposition, previousincidents with cancer, and pre-existing non-cancer diseases.

The term “subject suspected of having PTC” as used herein, refers to asubject that presents one or more symptoms indicative of PTC or that isbeing screened for PTC (e.g., during an examination). A subjectsuspected of having PTC may also have one or more risk factors. The termencompasses individuals who have not been tested for PTC, individualswho have received an initial diagnosis (e.g., a CT scan showing a mass)but for whom the stage of cancer is not known, as well as individualsfor whom the stage and/or grade of cancer has been determined by aconventional method (e.g., Gleason score). The term also includespatients who have previously undergone therapy for PTC.

The term “subject diagnosed with PTC” as used herein, refers to asubject who has been tested and found to have PTC. The diagnosis may beperformed using any suitable method, including, but not limited to,biopsy, x-ray, blood test, and the methods of the present invention.

The term “subject” or “patient” as used herein, refers to any mammal ornon-mammal that would benefit from determining the benefit fromtreatment, treatment, diagnosis, therapeutic monitoring, and/orprognosis. In certain examples a subject or patient includes, but is notlimited to, humans, farm animals (cows, sheep, pigs, and the like),companion animals (such as cats, dogs and horses, and the like),non-human primates and rodent (such as mice and rats). In a specificembodiment, the subject is a human.

The identification of a subject having increased likelihood ofdeveloping PTC or having metastatic PCT or recurrent (PTC), indicatesthat such a subject is a candidate for treatment of such metastatic orrecurrent PTC.

The term “treatment” as used herein, refers to clinical intervention inan attempt to alter the course of the subject or cell being treated. Innon-limiting examples, treatment includes preventing or delayingrecurrence of disease, alleviation of symptoms, diminishment of anydirect or indirect pathological consequences of the disease, preventingmetastasis, decreasing the rate of disease progression, amelioration orpalliation of the disease state, and remission or improved prognosis. Insome examples such treatment decreases the risk of local and regionalrecurrence and improves quality-of-life of said subject.

Treatment of papillary thyroid carcinoma includes, at a minimum, totalthyroidectomy with a possible lymph node dissection in the centralcompartment of the neck to remove local metastatic deposits. Thedecision to proceed with lymph node dissection is at the discretion ofthe surgeon and depends on the preoperative investigation includingultrasound and clinical examination. Following surgery radioactiveiodine is given to patients especially those patients with tumors largerthan 1.5 cm per those patients with concerning features on pathologyincluding signs of vascular and lymphatic invasion in the tumorspecimen. External beam radiation therapy may be used in a smallpercentage of cases if the papillary thyroid carcinoma is extremelyaggressive and it is invading outside of the thyroid and involving themuscles in the neck or the trachea or esophagus. In cases of recurrentpapillary thyroid carcinoma all of the previous treatment modalitiesincluding surgery, radioactive iodine, external beam radiotherapy may beused depending on the size and site of recurrence and if it is amenableto surgical resection. In cases that fail these methods of treatmentclinical trials (for example Phase I clinical trials) are used to testthe possibility that tyrosine kinase inhibitors may slow the growth ofthe tumor.

In one example, treatment of metastatic PTC or recurrent PTC includes,but is not limited to, neck dissections (including prophylactic neckdissections) and/or adjuvant radioiodine therapy.

In one example, treatment of metastatic PTC or recurrent PTC includes,but is not limited to, administration of a pharmaceutical composition.In some examples, the pharmaceutical composition is a tyrosine kinaseinhibitor. In some examples, the pharmaceutical composition comprisessorafenib, sunitinib, axitimb, or motisanib.

Thus, in some examples, the methods as described herein are useful indetermining the benefit from treatment of a subject with cancer.

The term “cancer” as used herein, refers to or describes thephysiological condition in a subject, such as a mammal, that istypically characterized by unregulated cell growth

In some examples, the methods as described herein are useful indetermining the benefit of treatment of a subject with a cancer that isspread via the lymphatic system. Examples include, breast cancer, coloncancer, and PTC. In a specific example said cancer is PTC.

The term “determining the benefit from treatment” as used herein,generally refers to assessing whether a patient is a suitable candidatefor treatment. The patient may be at risk of having cancer (such asPTC), or suspected of having cancer (such as PTC), or has been diagnosedwith cancer (such as PTC), or has an increased likelihood of developingmetastatic cancer (such as PTC). In some examples, a patient which isdetermined to benefit from treatment is a suitable candidate forsurgery, and/or radiation therapy (such as radioactive iodine ablation),and/or chemotherapy.

There is provided herein a method for identifying a subject withincreased likelihood of developing or having metastatic or recurrentcancer, comprising determining the presence of a prognostic marker in asample of said patient.

In another example, there is provided herein a method for identifying asubject with increased likelihood of developing or having metastatic orrecurrent papillary thyroid cancer (PTC), comprising determining thepresence of a prognostic marker in a sample of said patient.

In one example, a method as described herein comprises qualitatively orquantitatively determining, analyzing or measuring a biological samplefrom a subject for the presence or absence, or amount or concentration,of one or more prognostic marker (or biomarker) associated with thediagnosis and/or prognosis and/or therapeutic monitoring of metastaticcancer or recurrent cancer.

In a specific example, a method as described herein comprisesqualitatively or quantitatively determining, analyzing or measuring abiological sample from a subject for the presence or absence, or amountor concentration, of one or more prognostic marker (or biomarker)associated with the diagnosis and/or prognosis and/or therapeuticmonitoring of metastatic PTC or recurrent PTC.

The term “prognostic marker” or “biomarker” as used herein refers to amarker that informs about the outcome of a patient in the absence ofsystemic therapy or portends an outcome different from that of thepatients without the marker, despite empiric (not targeted to themarker) systemic therapy.

The term “prognosis” as used herein, refers to the prediction of thelikelihood of cancer-attributable death or progression, includingrecurrence, metastatic spread, and drug resistance, of a neoplasticdisease, such as PTC.

The term “diagnosis” as used herein, refers to the identification of amolecular and/or pathological state, disease or condition, such as theidentification of PTC, or other type of cancer.

The term “therapeutic monitoring” as used herein refers to theobservation of the response of the subject to the treatment administeredto it.

The determination, analysis or measurement of the biomarker iscorrelated with the benefit of treatment of PTC in the patient. In someexamples, a patient sample is compared to a control sample. In someexamples, a control is not used and qualitative or quantitative methodsare used to determine the presence or absence, or amount orconcentration of the protein of interest.

The term “sample” as used herein, encompasses a variety of cells,cell-containing bodily fluids and/or secretions as well as tissuesincluding, but not limited to a cell(s), tissue, whole blood,blood-derived cells, plasma, serum, sputum, mucous, bodily discharge,and combinations thereof, and the like.

Methods of obtaining such samples from a subject are known to theskilled worker.

As used herein, “obtaining a sample” or “obtaining a biological sample”refers to such methods as will be well known to the skilled worker. Abiological sample may be obtained directly or indirectly from thesubject. The term “obtaining” a biological sample may comprise receivinga biological sample from an agent acting on behalf of the subject. Forexample, receiving a biological sample from a doctor, nurse, hospital,medical center, etc., either directly or indirectly, e.g. via a courieror postal service. In some cases the biological sample is obtained fromarchival repositories. In one example, the methods of the invention arecarried out in vitro or ex vivo.

Means for enriching for cancer cells in a sample are known in the art.For example, the tissue may be isolated from paraffin or cryostatsections. Cancer cells may also be separated from normal cells by flowcytometry or laser capture microdissection. These, as well as othertechniques for separating cancerous from normal cells, are known in theart.

In one example, and in the case of PTC, a sample is obtained using fineneedle aspirate (FNA).

In one example, in determining whether there is strong, moderate orminimal (or absent) amount of the biomarker, the patient sample may becompared to one or more control samples. In one example, a controlsample has had known and/or established level of the biomarker. In oneexample, a control sample is a patient sample that has known and/orestablished levels of biomarker expression and/or known clinicaloutcome. In one example, a control is a cell line that has a knownamount of biomarker expression.

The term “expression”, as used herein, and for example in reference to abiomarker such as PDGFR-α, refers to all indicators of transcriptionalexpression of the biomarker encoding gene. Such indicators includebiomarker transcript products, generated as a result of transcription ofthe biomarker gene; translation products, including all forms of thebiomarker protein, generated as a result of translation of the biomarkertranscripts; and demonstrable or otherwise measurable biomarkeractivity.

As used herein, “biomarker protein”, includes, but is not limited to,full-length proteins, mature proteins, pre-proteins, polypeptides,isoforms, mutations, variants, post-translationally modified proteinsand variants thereof. Biomarker protein detection is know to the skilledworker, and is discussed herein.

Biomarker transcripts or mRNA can be measured using any of manytechniques known to those of skill in the art, including, but notlimited to, northern hybridization, PCR, reverse transcription followedby PCR, quantitative real-time PCR, nuclease protection assay, and insitu hybridization.

Biomarker activity can be measured by a variety of assays known to thoseof skill in the art. A suitable method can be selected to determine theactivity of proteins encoded by the biomarker genes according to theactivity of each protein analyzed. For biomarker proteins, polypeptides,isoforms, mutations, and variants thereof known to have enzymaticactivity, the activities can be determined in vitro using enzyme assaysknown in the art. Such assays include, without limitation, proteaseassays, kinase assays, phosphatase assays, reductase assays, among manyothers. Modulation of the kinetics of enzyme activities can bedetermined by measuring the rate constant K_(M) using known algorithms,such as the Hill plot, Michaelis-Menten equation, linear regressionplots such as Lineweaver-Burk analysis, and Scatchard plot.

Biomarker protein can be measured/detected by a variety of techniquesknown to the skilled worker, including, but not limited to, immunoassaysusing a biomarker specific antibody. Protein levels can also bedetermined using a specific antibody or mass spectroscopy in conjunctionwith 2 dimensional gel electrophoresis (separation of proteins by theirisoelectric point (IEF) in the first dimension followed by molecularweight determination using sodium dodecyl sulphate polyacrylamide gelelectrophoresis (SDS-PAGE)).

In other examples, a biomarker protein is detected using a binding agentincluding, but not limited to, a lectin, nucleic acid (e.g. DNA, RNA),monoclonal antibody, polyclonal antibody, Fab, Fab′, single chainantibody, synthetic antibody, aptamer (DNA/RNA), peptoid, zDNA, peptidenucleic acid (PNA), locked nucleic acid (LNA), synthetic or naturallyoccurring chemical compound (including but not limited to a drug orlabeling reagent), dendrimer, or any combination thereof. In someinstances, a single agent is used to detect a biomarker. In otherinstances, a combination of different agents is used to detect abiomarker

Detection includes direct and indirect detection. Similarly, a bindingagent can be directly or indirectly labeled.

The quantity of one or more biomarkers can be indicated as a value. Thevalue can be one or more numerical values resulting from the evaluationof a sample, and can be derived, e.g., by measuring level(s) of thebiomarker(s) in a sample by an assay performed in a laboratory, or fromdataset obtained from a provider such as a laboratory, or from a datasetstored on a server.

In some examples, qualitatively or quantitatively determining, analyzingor measuring a biological sample from a subject for the presence orabsence, or amount or concentration, of one or more prognostic markerassociated, is carried out using antibodies to the biomarker.

In a specific example, antibodies of the present invention areimmunoreactive or immunospecific for, and therefore specifically andselectively bind to a biomarker, for example the protein PDGFRα. In oneexample, antibodies which are immunoreactive and immunospecific forPDGFRα can be used. Antibodies PDGFRα are preferably immunospecific.

The term “antibody” and “antibodies” includes, but is not limited to,monoclonal and polyclonal antibodies. Antibodies may be derived frommultiple species. For example, antibodies include rodent (such as mouseand rat), rabbit, sheep, camel, chicken, and human antibodies. Inanother example, antigen binding fragments which specifically bind toPDGFRα are used. In some example, the antibodies also comprise a label.

The term “label” as used herein is an identifiable substance that isdetectable in an assay and that can be attached to a molecule creating alabeled molecule. The behavior of the labeled molecule can then bemonitored and/or studied and/or detected.

Examples of labels include, but are not limited to, various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, radioactive materials, positron emittingmetals using various positron emission tomographies, and nonradioactiveparamagnetic metal ions. The detectable substance may be coupled orconjugated either directly to the antibody (or fragment thereof) orindirectly, through an intermediate. The particular label used willdepend upon the type of immunoassay. Antibodies can be tagged with suchlabels by known methods.

The term “binds specifically” refers to high avidity and/or highaffinity binding of an antibody to a specific polypeptide e.g., anepitope of PDGFR-α. Antibody binding to its epitope on this specificpolypeptide is stronger than binding of the same antibody to any otherepitope, particularly those which may be present in molecules inassociation with, or in the same sample, as the specific polypeptide ofinterest. Antibodies which bind specifically to a polypeptide ofinterest may be capable of binding other polypeptides at weak, yetdetectable, level. Such weak binding, or background binding, is readilydiscernable from the specific antibody binding to the compound orpolypeptide of interest, e.g., by use of appropriate controls, as wouldbe known to the worker skilled in the art.

In one example, a sample containing cancerous cells or suspected ascontaining cancerous cells is obtained from the patient which is at riskfor PTC, is suspected of having PTC, and/or has been diagnosed with PTC.Collection of such a sample is well known to the skilled worker. In aspecific example, the sample is a fine needle aspirate (FNA) sample.Methods of obtaining a FNA sample, processing and/or storage of such asample are also well known to the skilled worker. In other example, asample is obtained from surgical dissection.

Tissue samples may be fresh-frozen and/or formalin-fixed,paraffin-embedded tissue blocks prepared for study byimmunohistochemistry (IHC). In one example, the sample is a formalinfixed and/or paraffin-embedded tumor tissue from a biopsy or surgicalresection of a cancer (e.g., tumour). Samples may also be processed by,snap freezing, treated to isolate DNA, RNA, or protein, or anycombination.

The methods of the present invention may be accomplished using anysuitable method or system of immunohistochemistry or quantifying levelsof mRNA. Non limiting examples include automated systems, quantitativeIHC, semi-quantitative IHC, RT-PCR and qRT-PCR and manual methods.

The term “quantitative” immunohistochemistry refers to an automatedmethod of scanning and scoring samples that have undergoneimmunohistochemistry, to identify and quantitate the presence of aspecified biomarker, such as an antigen or other protein. For example,to quantitate PDGFR-α, the score given to the sample is a numericalrepresentation of the intensity of the immunohistochemical staining ofthe sample, and represents the amount of target biomarker present in thesample. As used herein, Optical Density (OD) is a numerical score thatrepresents intensity of staining as well as the percentage of cells thatare stained. As used herein, semi-quantitative immunohistochemistryrefers to scoring of immunohistochemical results by human eye, where atrained operator ranks results numerically (e.g., as 0, 1 or 2).

In a specific example, expression of the biomarker in a sample isassessed by an operator as “2+” (denoting strong staining), “1+”(denoting moderate staining), or “0” (denoting minimal or absentstaining). In some instance, each sample is assessed in duplicate, ortriplicate. In another specific example, Nuclear staining is not scoredand the correlations for staining were assessed using Fisher's exacttest for tables and Spearman rank correlation for continuous variables.

In one example of the methods described herein, a biological sample froma subject is assessed for presence of a biomarker within the biologicalsample, wherein the levels and/or concentration of the biomarkerindicates the aggressiveness or metastatic potential of PTC.

Automated sample processing, scanning and analysis systems suitable foruse with immunohistochemistry are known in the art, and may be used withthe methods described herein. Such systems may include automatedstaining and microscopic scanning, computerized image analysis, serialsection comparison (to control for variation in the orientation and sizeof a sample), digital report generation, and archiving and tracking ofsamples (such as slides on which tissue sections are placed). Cellularimaging systems are commercially available that combine conventionallight microscopes with digital image processing systems to performquantitative analysis on cells and tissues, including immunostainedsamples.

In a specific example, the detection, analysis or measurement of PDGFR-αprotein within a tissue sample is carried out using immunohistochemistry(IHC). It will be clear to the skilled worker that other immuno assays,both qualitative and quantitative, may be used in the present invention.

In one example, immunohistochemistry is carried out using tissuemicroarrays from formalin tissues.

Other examples that may be used in the detection, analysis ormeasurement of PDGFR-α include, but are not limited to,immunoprecipitation, immunoblotting, mass spectrometry, quantitativefluorescence activated cell sorting, enzyme linked immunosorbent assay,immunohistochemistry, quantitative immunohistochemistry, fluorescenceresonance energy transfer, Forster resonance energy transfer, andbiomolecular fluorescence complementation.

In determining whether there is strong (e.g., 2+) or moderate (e.g., 1+)or minimal (e.g., 0) PDGFR-α staining, the patient sample may becompared to one or more control samples. In one example, a controlsample is a patient sample that has known and/or established levels ofPDGFR-α tumour staining and/or known clinical outcome. In one example, acontrol is a cell line that has a known amount of PDGFR-α staining.

In some example, a control is not used and qualitative or quantitativemethods are used to determine the level of staining.

In practice, in the example in which a patient sample is determined tohave moderate (1+) or strong (2+) expression (i.e., strong expression ofPDGFR-α), the patient is identified as a subject with an increasedlikelihood of developing or having metastatic papillary thyroid cancer(PTC), or a subject with an increased likelihood of developing or havingrecurrent PTC, and so is considered a good candidate for, and subjectedto treatment comprising, surgical resection and/or radio therapy (suchas radio iodine ablative therapy or external beam radiotherapy), and/orchemotherapy.

In practice, in the example in which a patient sample is determined tohave minimal staining (“0”) expression (e.g., absent or minimalexpression of PDGFR-α), the patient is identified as a subject nothaving an increased likelihood of developing or having metastaticpapillary thyroid cancer (PTC), or a subject not having an increasedlikelihood of developing or having recurrent PTC. Continued treatmentoptions for such patients identified as not having a likelihood ofdeveloping or having metastatic or recurrent are known to the skilledworker. For example, patients lacking confirmed metastases from PTC inmany cases do not get radioactive iodine, but this depends on the sizeof the primary tumor. Regardless of their initial treatment includingsurgery and/or radioactive iodine all patients are followed with serialimaging including whole body iodine scans, neck ultrasound and serumthyroglobulin assessments on a yearly basis essentially for the rest oftheir life although the interval between follow-up visits decreases fromsix months initially for approximately 2 years to yearly after that.Metastases from thyroid cancer typically occur within 2 to 3 years thatcan come back as late as 10 or 15 years after their original surgery andtreatment.

In a specific example, in the case of examined PDGFR-α and p expressionin a tissue array of papillary thyroid cancer including primary tumorspecimens with (n=58) and without (n=66) nodal metastases, for PDGFRα,in primary tumors without lymphatic metastases, a small fraction of thetumors were positive (16%) but most of the staining was moderate at1+(Table 2). However, in primary tumors with lymph node metastases themajority (83%) of the tumors were positive for PDGFRα expression(p=0.003). Nodal deposits in all but one case are positive for PDGFR-αwith most cases exhibiting strong staining (Table 2). PDGFR-β stainingin primary tumor specimens demonstrated significantly different results.PDGFR-β staining did not follow a pattern with respect to the absence orpresence of nodal metastases. Approximately 90% of all tumors, a nearlyequal fraction of lymph node negative and lymph node positive cases,stained for PDGFR-β and staining qualitatively was also very similar(p=0.82) (see Table 2).

In another specific example, in the case of freshly prepared PTC tumourisolated at operative resection, with and without nodal metastases,PDGFR (α and β) configuration and nodal involvement was determined in 14cases that included level 6 lymph node dissections as shown in FIG. 2.Only 2 of 7 primary tumors without nodal metastases expressed PDGFR-α(patient #5 and #7). In fact the only clearly positive result expressingPDGFR-α (patient #7) was a false positive due to an unexpected case ofsarcoidosis with fibrotic reactions in all of the nodes removed asdocumented clearly on pathology.^(47,48) In 7 of 7 primary tumors withnodal metastases we observe PDGFR-α expression. Even if we include thelikely false positive (patient #7) and the very weak staining in patient#5, the difference in number of positive cases for PDGFR-α between node−(2/7) and node+ (7/7) cases is significant (P=0.02). All of the nodalmetastases examined express PDGFR-α although the levels varysignificantly (FIG. 2).

It will be appreciated that in some circumstances, a patient which isinitially identified a not having an increased likelihood of developingor having metastatic PTC or recurrent PTC, may relapse or reoccur. Sucha reoccurrence can manifest is several ways, including but not limitedto, reoccurrence of the primary tumour and development of metastasis. Inaddition to, or alternatively, an additional distinct tumour can arise.The methods as described herein may be used in the therapeuticmonitoring of a patient, to monitor and identify those patients whichmay relapse.

In accordance with one aspect of the present invention, there isprovided a method comprising: a) obtaining a sample from a subject with,or suspected as having, PTC; b) contacting the sample with an antibodyto a biomarker, to form a complex between the antibody and the biomarkerpresent in the sample; c) measuring the complex formed to determine theamount or concentration of said biomarker in the sample; wherein thedetermination of a benefit for treatment is determined by a strongexpression of the biomarker in the sample. In one example, the biomarkeris PDGFRα. In one example, expression of the biomarker in the sample iscompared to a control. In one example, said sample comprises a FNAsample.

In another embodiment, PDGFRα expression is associated with thedevelopment of metastasis in a patient with PTC.

In accordance with another aspect of the present invention, there isprovided a method comprising: a) obtaining a sample from a subject with,or suspected as having, PTC; b) processing said sample, c) contactingthe processed sample with an analyte or reagent to a biomarker, to forma complex between the analyte or reagent and the biomarker present inthe processed sample; c) measuring the complex formed to determine theamount or concentration of said biomarker in the sample; wherein thedetermination of a benefit for treatment is determined by a high amountor concentration (or strong expression) of the biomarker in the sample.In one example, the biomarker is PDGFRα. In one example, expression ofthe biomarker in the sample is compared to a control. In one example,said sample comprises a FNA sample. In another embodiment, PDGFRα isassociated with the development of metastasis in a patient with PTC.

There is further provide the step of treating a subject with a highconcentration or amount (or strong expression) of PDGFRα with atreatment for PTC.

In some examples, processing said sample permits analysis of thebiomarker within the sample. In some example, processing refers toisolating or extracting biomarker transcript from said sample, said RNAsuitable for analysis. In some examples, processing refers to isolatingof extracting biomarker protein from said sample, said protein suitablefor subsequent analysis.

Analysis includes, but is not limited to, performing an analyte assaywith said processed sample. For example, performing an analyte bindingassay configured to detect a biomarker in said processed tumor sample byintroducing the processed tumor sample into an assay instrument which(i) contacts a reagent which specifically binds for detection of thebiomarker within the tumor sample, and (ii) generates one or more assayresults indicative of binding of said biomarker. As noted herein, saidbiomarker is a biomarker protein, a biomarker transcript, or biomarkeractivity.

In some examples, the analyte binding assay in an immunoassays. In someexamples, said immunoassay is immunohistochemistry. In some example saidimmunohistochemistry is performed with an automated system, or a manualsystem.

In some example, the analyte binding assay comprises detecting saidbiomarker RNA, include but limited to, using RT-PCT or in situhybridization.

The assay results may be quantitative or semi-quantitative.

Additional specific examples of processing comprises formalin fixing, orparaffin-embedding, or both formalin fixing and paraffin-embedding, saidsample.

Treatment of said subject may comprise surgical resection of thyroid andlymph nodes, radio therapy, chemotherapy, or combinations thereof. Radiotherapy comprises radio iodine ablative therapy or external beamradiotherapy. Chemotherapy comprises a tyrosine kinase inhibitor such assorafenib, sunitinib, axitimb, or motisanib.

Treatment may comprise administering an inhibitor of PDGFR-α to saidsubject. In some examples said inhibitor comprises, an RNA interferencemolecule, a small molecule, nucleic acid, an antibody, a peptide, apharmaceutical composition, an aptamers, or combinations thereof. Insome examples said RNA interference molecule comprises a RNAi molecule,a siRNA molecule, or a shRNA molecule.

The methods described herein are useful in the modulation of PTCprogression.

As used herein, the term “modulation of PTC progression” refers to theability of a compound to increase or decrease the likelihood that a PTCwill progress to an aggressive prostate cancer and/or will metastasize.Generally, compounds therapeutically useful are those that decrease thelikelihood of PTC progression.

Accordingly, in one example, a subject identified with a likelihood ofdeveloping or having metastatic PTC is treated so as to modulated PTCprogression, and in particular to decrease the likelihood of PTCprogression. In one example, inhibition of PDGFRα reduces the likelihoodof a patient with PTC developing metastases. In a specific example, asubject identified with a likelihood of developing or having metastaticPTC is treated with an inhibitor of PDGFRα.

There is provided a method for the treatment of a subject with alikelihood of developing or having metastatic PTC, comprisingadministering to said subject an inhibitor of PDGFRα.

Inhibitors of PDGFRα include, but are not limited to, RNA interferencemolecules, small molecules, nucleic acids, antibodies, peptides,pharmaceutical compositions, and/or aptamers.

Examples of RNA interference molecules include a RNAi molecule, a siRNAmolecule, or a shRNA molecule.

The term siRNA (short interfering RNA) or siRNA duplexes, as used hereinhas the same meaning as typically in the art. i.e. the term siRNA refersto double stranded RNA complex. Often, the complex has 3′-overhangs. Inone example, siRNA are commercially available.

Pharmaceutical compositions include, but are not limited to, sorafenib,sunitinib, axitimb, or motisanib.

A compound or composition may be administered alone or in combinationwith other treatments, either simultaneously or sequentially.

Methods of the present invention are conveniently practiced in the formof a kit. Such a kit preferably contains antibodies for PDGFRα andinstructions for the use thereof. In a specific example, the kit furthercomprises at least one control sample for PDGFRα.

As described herein, there is provided a kit for identifying a subjectwith an increased likelihood of developing or having metastaticpapillary thyroid cancer (PTC), or a subject with an increasedlikelihood of developing or having recurrent PTC, comprising: a)instructions for determining the amount of PDGFRα in a sample from saidpatient; b) a reagent for measuring the amount PDGFRα in said sample,wherein in the case in which said patient sample is determined to have“2+” or strong expression (i.e., strong expression of PDGFR-α), saidpatient is identified as a subject with an increased likelihood ofdeveloping or having metastatic papillary thyroid cancer (PTC), or asubject with an increased likelihood of developing or having recurrentPTC. In one example, said reagent is an antibody to PDGFRα. In oneexample positive and/or negative control samples are also included inthe kit.

As described herein, there is provided systems for treating a subjectwith or suspected of having papillary thyroid cancer, comprising: a) areagent which specifically binds for detection of PDGFR-α in a tumorsample from a patient with thyroid cancer, and b) an assay instrumentconfigured to receive a tumor sample and contact the reagent with thetumor sample, and to generate one or more assay result indicative ofbinding said reagent with the PDGFR-α within the tumor sample which isassayed for specific binding.

To gain a better understanding of the invention described herein, thefollowing examples are set forth. It should be understood that theseexamples are for illustrative purposes only. Therefore, they should notlimit the scope of this invention in anyway.

EXAMPLES Example I Patients and Methods

Patient Specimens

Ethics approval was obtained through the University of Alberta HeathResearch Ethics Board ID Pro00018758. Specimens prepared for primarycell culture were placed in culture media within 10 minutes ofdevascularisation. For tissue banking specimens were placed in OCT(Optimal Cutting Temperature compound) within 20 minutes ofdevascularisation and snap frozen in liquid N₂. For the tissue arraywith paraffin specimens, a total of 124 patients were selected withpapillary thyroid carcinoma, 66 without and 58 with lymphaticmetastases. In all cases patients had a total thyroidectomy with a levelVI lymph node dissection such that histopathology could be used todocument the true node negative cases that complemented our clinicalassessment via ultrasound. Both primary tumor specimens and matchingnodal metastases were available in 13 cases. Cases included were allpapillary thyroid carcinoma in the absence of any aggressive variantssuch as insular or tall cell papillary thyroid cancer. Two pathologistsseparately assessed the specimens to document primary tissue diagnosisas well as the presence of lymphatic metastases in nodes sectioned.

Immunohistochemistry

Immunohistochemistry was performed using standard techniques. Briefly,formalin-fixed, paraffin-embedded tissue sections of 4 μM thickness weredeparaffinized and rehydrated. The antibodies used for the Western blotsand for staining the paraffin tissue arrays as follows: antibodiesagainst Akt, phospho-Akt (Ser473), PDGFR-α, phospho-PDGFR-α/β (Tyr849)/β(Tyr857), p44/42MAPK/ERK, and phospho-p44/42 MAPK/ERK (Thr202/Tyr204)were all purchased from Cell Signalling Technology (Danvers, Mass.,USA). The PDGFR-β antibody was purchased from Santa Cruz Biotechnology,(Santa Cruz, USA). Heat-induced epitope retrieval was performed usingcitrate buffer (pH 6.0) and pressure cooked in a microwave for 20minutes. The endogenous peroxidase activity was blocked using 3% H₂O₂ inmethanol for 10 minutes. Tissue sections were then incubated with ThePDGFR-α and -β overnight at 4° C. in a humidified chamber. After 2washes with PBS, tissue slides were incubated with biotinylated linkeduniversal secondary antibody and subsequently with streptavidin-HRPcomplex as per the manufacturer's instructions (LSAB+ system, Dako).Tissue sections were incubated with 3,3′-diaminobenzidine/H₂O₂ (Dako)for color development and counter-stained with hematoxylin.

Marker Scoring and Statistical Analysis

Evaluation of immunostaining was performed without knowing the clinicaloutcome and the other staining results. The cytoplasmic expression ofPDGFR-α and PDGFR-β was assessed for each case, in triplicate, as 2+(strong staining), 1+ (moderate staining), 0 (minimal staining). Nuclearstaining was found in all specimens and not scored. The correlations forstaining were assessed using Fisher's exact test for tables and Spearmanrank correlation for continuous variables. Sample cores on the tissuearray that were fragmented or incomplete were not scored.

Cell Culture

TPC-1, KTC-1, BCPAP experimental cell lines were all generously providedby Dr. Ezzat, University of Toronto. 8305C was purchased from DSMZ(Braunschweig, Germany). RET/PTC, BRAF and RAS mutation status asoutlined in Table 1 and cell origin confirmed using Pax-8 and TTF-1staining (Table 1).⁴⁵ RPMI 1640 was purchased from Life Technologies(Grand Island, N.Y.). Standard fetal bovine serum (FBS) was purchasedfrom Hyclone (Logan, Utah, USA). Trypsin-EDTA containing 0.25% trypsin,and PDGF-BB were purchased from GIBCO (Invitrogen, Grand Island, N.Y.,USA). Sunitinib malate was purchased from TORCIS Bioscience (EllisvilleMo. USA).

Primary cell culture and experimental cell lines were maintained in RPMI1640 media supplemented with 10% FBS. The cells were seeded in a 100-mmculture dish and were grown in a humidified 5% CO₂ incubator. ForPDGF-BB stimulation (25 ng/ml), cells were grown to about 80% confluenceand incubated in serum-free medium overnight prior to each experiment.MAPK/ERK inhibitors U0126 and PD98059 were purchased from Calbiochem(Toronto, Ontario, Canada). PI3K/Akt inhibitor ly294002 was purchasedfrom Cell Signaling Technology (Danvers, Mass., USA). Cells that weretreated with inhibitors were given varying concentrations: U0126; 2umol/L and 10 umol/L; Ly294002 10 umol/L and 25 umol/L; Sunitinib 0.25umol/L. In all cases, unless otherwise indicated the inhibitors weregiven to cells and 60 minutes later the PDGF-BB was added to thecultures followed by Western blot analysis.

Western Blot Analyses

Cells were lysed in RIPA buffer [150 mM NaCl, 100 mM Tris (pH 8.0), 1%Triton X-100, 1% deoxycholic acid, 0.1% SDS, 5 mM EDTA, and 10 mM NaF]supplemented with 1 mM sodium vanadate, 2 mM leupeptin, 2 mM aprotinin,1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM DTT, 2 mM pepstatin, and1:100 protease inhibitor cocktail set III on ice. After centrifugationat 4° C. at 18,000 rpf for 15 min, the supernatant was harvested as thetotal cellular protein extracts and stored at −80° C. The proteinconcentration was determined using Bio-Rad protein assay reagent(Richmond, Va., USA). Running samples were prepared by adding a samplereducing agent and SDS sample buffer, incubating at 98° C. for 5 min.Aliquots of protein extract samples were separated by SDS-polyacrylamidegel electrophoresis (SDS-PAGE) and transferred to nitrocellulosemembrane. Membranes were blocked with 5% nonfat dry milk in 1×TBScontaining 0.05% Tween-20 for 60 min, followed by incubation withprimary antibodies 4° C. overnight. Protein bands were detected byincubation with horseradish peroxidase-conjugated antibodies (PierceBiotechnology, Rockford, Ill., USA) and visualized with SuperSignal WestPico chemiluminescence substrate (Thermo Scientific, Rockford, Ill.,USA).

Short Interfering RNA (siRNA) and Transfections

siRNA for PDGFR-α and scrambled siRNA were purchased from Siegen(Foster, Calif., USA). Transient transfections of TCP-1, 8305C cells(3×10⁶ cells) were performed using the Electro square electroporator BTXECM 800 (225V, 8.5 ms, 3 pulses). 1 nmol/L of siRNA or scrambled controlwas used 3 million of TPC-1 and 8305C cells. The efficiency of targetgene inhibition was assessed after 48 hours transfection by usingWestern blotting.

Transwell Migration/Invasion and Cell Growth Assays

As previously described, Cytoselect™ 96-well cell invasion assay kit wasused (Cell Biolabs, San Diego, Calif., USA) to assess cell invasivenessaccording to the manufacturer's protocol.⁴⁶ Briefly, TPC-1, BCPAP and8305C after transfected with either PDGFRα siRNA or scramble siRNA,10000 cells were starved overnight and added to the inserts, PDGF-BB wasadded to some wells at the concentration of 25 ng/mL, PDGF-BB was added1 h later for inhibitors, sunitinib (0.2 umol/L), Ly294002 (10 umol/L)and (U0126) 2 umol/L, cells were seeded into the insert plate for 16hours, the invasive cells passed through basement membrane layer to thebottom and dissociated from membrane by the addition of cells detachmentbuffer, the invasive cells were lysed and followed by quantificationusing CyQuant GR fluorescent Dye. For cell viability analysis 10000cells were seeded per well and cultured for 16 hours. MTS assay(Promega, Madison, USA) was then performed in 4 replicates followedmanufacturer's instructions. PTC cell lines transfected with PDGFRspecific siRNA or scrambled control were plated at a density of 10,000or 20,000/ml and cultured for 5 days. Cell counts were done on days 2, 3and 5 using trypan blue (Sigma-Aldrich, Oakville, Canada) and resultsexpressed as total number of viable cells. MTS assay was done in 7replicates as per manufacturer's instructions. The absorbance wasrecorded by a BioRad spectrophotometer at day 5 of cell culture.

Statistical Analysis

Data were expressed as the mean±S.E. from a minimum of three independentexperiments. Statistical analyses were performed with a completelyrandom design one-way ANOVA. The correlations between PDGFR and theother biological markers were assessed using Fisher's exact test fortables and Spearman rank correlation for continuous variables.Statistical tests are two-tailed with a P value <0.05 considered to bestatistically significant. The SAS computer program SAS (r) 9.2 (TS1M0)was used to perform the analysis

Results

PDGFR-α Expression, but not PDGFR-β, is Associated with LymphaticMetastases in Papillary Thyroid Cancer

We examined PDGFR-α and β expression in a tissue array of papillarythyroid cancer including primary tumor specimens with (n=58) and without(n=66) nodal metastases with representative sections shown in FIG. 1.FIG. 1 shows representative immunohistochemical stains of PDGFR-α inpapillary thyroid cancer (PTC) primary tumors without nodal metastasesat low power (A) (100×) and high power (B) (400×). Primary tumors withnodal metastases are shown (C) (100×) and (D) (400×). Cytoplasmicstaining demonstrates much higher levels of PDGFR-α in node-positive (Cand D) primary tumor specimens as opposed to node-negative specimens (Aand B).

All patients had a level 6 lymph node dissection to best assess thepossibility of lymphatic metastases. Included in the array wereneighboring, normal thyroid tissue cores (n=32) and matching nodalthyroid cancer metastases from 13 primary tumors. For PDGFR-α, inprimary tumors without lymphatic metastases, a small fraction of thetumors were positive (16%) but most of the staining was weak at 1+(Table 2). However, in primary tumors with lymph node metastases themajority (83%) of the tumors were positive for PDGFR-α expression(p=0.003) (Table 2). Moreover, nodal deposits in all but one case arepositive for PDGFR-α with most cases exhibiting strong staining (Table2). PDGFR-β staining in primary tumor specimens demonstratedsignificantly different results. PDGFR-β staining did not follow apattern with respect to the absence or presence of nodal metastases.Approximately 90% of all tumors, a nearly equal fraction of lymph nodenegative and lymph node positive cases, stained for PDGFR-β and stainingqualitatively was also very similar (p=0.82) (see Table 2). Nodalmetastases also commonly expressed PDGFR-β (>90%). Expression of PDGFR-αand -β was undetectable in most (95%+) of the non-neoplastic, normalthyroid tissue (Table 2).

TABLE 2 Percentage (%) of specimens staining for PDGFR-α and -β innormal thyroid, papillary thyroid cancer primary tumors and nodalmetastases. Scoring Stain 0 1+ 2+ PDGFR-α benign thyroid (n = 35) 97 3 0node negative primary (n = 66) 84 12 4 node positive primary (n = 58) 1739 44 lymph node metastasis (n = 13) 8 23 69 PDGFR-β benign thyroid (n =35) 94 6 0 node negative primary (n = 66) 13 30 57 node positive primary(n = 58) 11 42 47 lymph node metastasis (n = 13) 8 8 84

To further assess differences in PDGFR-α and -β expression, we examineda cohort of freshly prepared PTC tumors isolated at operative resection,with and without nodal metastases. PDGFR (α and β) configuration andnodal involvement was determined in 14 cases that included level 6 lymphnode dissections as shown in FIG. 2.

FIG. 2 shows Western blots of PDGFR-α and -β in patient primary tumorslacking nodal metastases (#1-7, far left) compared to those with nodalmetastases (#8-14, middle section). Corresponding metastatic diseasedeposits from patients #8-14 are shown in the far right section. PDGFR-αis expressed primarily in primary tumors with metastatic disease andcorresponding metastatic deposits (P=0.007). In contrast, PDGFR-β statusdoes not correlate with metastatic disease. Patient #7 has sarcoidosiswhich induces nodal proliferation and is a benign cause for increasedlevels of PDGFR-α. The analysis was completed in all cases from freshlyprepared tumor specimens. TPC-1 included as an internal control.

Only 2 of 7 primary tumors without nodal metastases expressed PDGFR-α(patient #5 and #7). In fact the only clearly positive result expressingPDGFR-α (patient #7) was a false positive due to an unexpected case ofsarcoidosis with fibrotic reactions in all of the nodes removed asdocumented clearly on pathology.^(47,48) In 7 of 7 primary tumors withnodal metastases we observe PDGFR-α expression. Even if we include thelikely false positive (patient #7) and the very weak staining in patient#5, the difference in number of positive cases for PDGFR-α between node−(2/7) and node+ (7/7) cases is significant (P=0.02). All of the nodalmetastases examined express PDGFR-α although the levels varysignificantly (FIG. 2). Similar to what we observed in the tissue array,PDGFR-β expression does not appear to be linked to metastases in thispatient cohort (FIG. 2). All but one of the specimens (patient #2)exhibited PDGFR-β staining but again the levels vary significantlybetween cases.

PDGFR-α Activation is Associated with MAPK/ERK and PI3K/Akt SignalingPathways

Current models for receptor tyrosine kinase signalling involve both theMAPK/ERK and PI3K/Akt pathways.³⁹ To assess the role of each of thesesignal transduction pathways in metastatic PTC, we used primary cellculture to examine MAPK/ERK and PI3K/Akt activation in PTC metastatictumour specimens.

FIG. 3 shows Western blot of primary cell culture obtained from a lymphnode specimen with metastatic papillary thyroid cancer confirmed byhistology. The results show activated PDGFR-α (phospho-PDGFR) whenstimulated with PDGF-BB with concomitant activation of the PI3K/Aktpathway as demonstrated by increasing levels of phospho-Akt. Thestimulatory effect can be blocked by sunitinib, a tyrosine kinaseinhibitor. Activation of the MAPK/ERK pathway (phospho-ERK) is presentbut to a much smaller degree in this semi-quantitative experiment.

Shown in FIG. 3 is a primary cell cultures of PTC metastatic tumourstimulated PDGF-BB with and without sunitinib to assess the effect ofTKI therapy on PDGFR activation and downstream signaling. We observedsignificant increases in phospho-Akt, consistent with activation of thePI3K/Akt pathway and a small increase in phospho-ERK (MAPK/ERK pathway)with PDGF-BB stimulation of these cultured tumor cells (FIG. 3).Activation of both pathways was completed blocked with the addition ofmultikinase inhibitor sunitinib. Having demonstrated the concept thatboth pathways may be activated in metastatic PTC, we examined the statusof both the MAPK/ERK and PI3K/Akt pathways in the 14 patients previouslyscreened for PDGFR-α and -β.

In FIG. 4, Western blots show the activation status of the MAPK/ERK andPI3K/Akt pathways in primary tumours lacking nodal metastases (#1-7, farleft) and those with nodal metastases (#8-14, middle section).Corresponding metastatic disease deposits from patients #8-14 are shownin the far right section. The analysis was completed in all cases fromfreshly prepared tumour specimens. TPC-1 cell line used internal controlfor the different membranes.

Shown in FIG. 4 is a representative Western blot documenting activationof both the MAPK/ERK and PI3K/Akt pathways in all of the PTC primarytumors, with and without metastases. Typically we observed that bothpathways are operative although in one case (patient #14) activation ofthe MAPK/ERK pathway was minimal.

PDGFR-α Activation Increases Invasive Potential in PTC Cell Lines andcan be Blocked with Tyrosine Kinase Inhibitors

Having demonstrated a strong correlation between PDGFR-α and nodalmetastases in clinical specimens, PTC experimental cell lines weresurveyed for differences in PDGFR receptor expression. We show here thatthere is a differential expression of PDGFR subtypes depending on thecell line (FIG. 5).

In FIG. 5, Western blot of PDGFR configuration in the papillary thyroidcancer cell lines is shown. TPC-1 is the only cell line with both alphaand beta subunits of PDGFR. The KTC-1 cell line represents an importantnaïve control for our experimental series. All cell lines expressPDGF-BB ligand. Cell line integrity and origin was confirmed usingthyroid-specific markers Pax-8 and TTF-1 (Table 1).

TPC-1 has both PDGFR-α and -β receptor isoforms, BCPAP has only PDGFR-β,8305C only exhibits PDGFR-α and the KTC-1 cell line does not have eithersubunit. Using the Cytoselect™ invasion assay and in the presence ofPDGFR ligand PDGF-BB, known to bind all subunits of PDGFR, wedemonstrate significant differences in invasion potential of the celllines depending on the configuration of the PDGFR. We also examined theeffect of TKI blockade on invasive potential in these cell lines. InPTC-1 and 8305C, two cell lines with PDGFR-α, PDGF-BB stimulation leadto a significant increase in invasive potential (FIG. 6).

Cytoselect assay results (in triplicate) for invasive potential of TPC-1(A), 8305C (B), and BCPAP (C) cell lines as shown in FIG. 6. The celllines with the PDGFR-α subunit (TPC-1 and 8305C) demonstrated increasedinvasive potential with PDGF-BB stimulation, but not the BCPAP linewhich has only PDGFR-β. Sunitinib virtually completely abates any changein invasive potential with PDGF-BB stimulation of PDGFR. Sunitinib doesnot alter invasive potential without PDGF-BB stimulation in any of thecell lines.

The addition of sunitinib, a multikinase inhibitor, abated any change ininvasive potential as a result of PDGF-BB stimulation for both celllines (FIG. 6). Conversely, the BCPAP cell line (PDGFR-β only) did notexhibit any change in invasive potential (FIG. 6). The effect ofsunitinib on cell growth for all of the cell lines in the absence ofPDGF-BB stimulation was not significant (FIG. 6). KTC-1 also did notexhibit any change in invasive potential with PDGF-BB stimulation andincreasing levels of PDGF-BB or longer periods of stimulation did notalter these results (not shown).

Selective Knockdown of PDGFR-α with siRNA Disrupts Invasive Potential

We used siRNA to disrupt expression of PDGFR-α and examined thesubsequent effect on the invasive potential of cell lines exhibitingPDGFR-α and -β (TPC-1) or only PDGFR-α (8305C).

Cytoselect invasion assays with or without PDGFR-α siRNA for TPC-1 cellline (A) with corresponding cell viability assessment (B) are shown inFIG. 7. Invasion assays for 8305C cell line with PDGFR-α siRNA shown in(C) with corresponding cell viability experiment (D). The resultsstrongly suggest that the PDGFR-α, but not -β, is essential to mediatingincreased invasive potential. siRNA significantly reduces PDGFR-αexpression as shown in the accompanying Western blot. RFU refers torelative fluorescence units.

Shown in FIG. 7 are invasion assays in TPC-1 transfected with PDGFR-αsiRNA (FIG. 7A) with corresponding cell viability assessments (FIG. 7B).Two different PDGFR-α siRNA constructs were nearly equally effective atdecreasing PDGFR-α protein expression in TPC-1 cells as shown in theinset Western blots (FIG. 7A). For the invasion assay in TPC-1 cellsusing PDGFR-α siRNA1, increases in invasive potential triggered byPDGF-BB stimulation were virtually completely disrupted (FIG. 7A).PDGFR-α siRNA alone did not significantly change invasive potentialrelative to the controls with scramble RNA. Cell viability was notaffected in any of the experiments (FIG. 7B). Qualitatively andquantitatively similar results were seen with siRNA blockade of PDGFR-αin 8305C cells (FIG. 7C). PDGFR-α siRNA could block PDGF-BB mediatedincreases in invasive potential but no effect was seen in the absence ofPDGF-BB. With or without PDGF-BB stimulation, scramble or siRNAconstructs had no effect on cell viability (FIG. 7D). PDGFR-α siRNA1construct was more effective at reducing PDGFR-α protein levels and wasused for these experiments in 8305C cells (inset FIG. 7C).

PDGFR-α Activation and Increased Invasion Potential is Mediated by Boththe MAPK/ERK and PI3K/Akt Pathways

Using Western blots we demonstrate a link between PDGFR activation andup-regulation of both the MAPK/ERK and PI3K/Akt pathways in TPC-1 and8305C cell lines. We also examine the impact of sunitinib treatment onsignal transduction in these cell lines.

The corresponding Western blots of the TPC-1 (A) and 8305C (B) celllines for the invasion assay as shown in FIG. 6, is presented in FIG. 8.Both TPC-1 and 8305C specimens show activation of PDGFR-α(phospho-PDGFR) with corresponding up-regulation of both the PI3K/Akt(phospho-Akt) and MAPK/ERK (phospho-ERK) pathways. Sunitinib completelydisrupts PDGFR activation and corresponding up-regulation of downstreamMAPK/ERK and PI3K/Akt pathways. The time-dependent changes in activationof PDGFR-α and downstream signaling pathways are shown for TPC-1 in (C).It is clear that the activation of the MAPK/ERK and PI3K/Akt pathways isessentially simultaneous.

Shown in FIG. 8 are the Western blots for (A) TPC-1 and (B) 8305C celllines examining activation of PDGFR as well as the MAPK/ERK and PI3K/Aktsignal transduction pathways. For both cell lines PDGF-BB stimulationleads to strong up-regulation phospho-PDGFR-α/β, phospho-Akt, andphospho-ERK. The degree of up-regulation for the MAPK/ERK pathway washigher in TPC-1 cells than in 8305C which exhibits a small increase inphospho-ERK (FIG. 8). This is likely due to the fact that the MAPK/ERKpathway in 8305C is constitutively activated with its known BRAFmutation (Table 1). It may indicate that activation of PI3K/Akt by BRAF,if operative, does not alter the responsiveness of this pathway to PDGFRsignaling. The addition of sunitinib, which we saw previously abatedchanges in invasive potential in TPC-1 and 8305C cell lines, completelyblocks activation of PDGFR-α/β with corresponding significant decreasedactivity in both the PI3K/Akt and MAPK/ERK pathways (FIG. 8). To confirmthat activation of the MAPK/ERK and PI3K/Akt pathways occurred on asimilar timescale, we stimulated TPC-1 cells with PDGF-BB and followedexpression of phospho-Akt and phospho-ERK over time (FIG. 8C). Wedemonstrate that PDGF-BB stimulation leads to virtually simultaneousincreases in phospho-PDGFR-α/β, phospho-Akt and phospho-ERK within 5minutes. Both pathways are maximally activated within 15 minutes andlevels slowly return to baseline near 60 minutes.

TABLE 1 Experimental papillary thyroid cancer cell lines Cell LinesHistology RET/PTC BRAF PAX8 TTF1 TPC-1 PTC + wt High Low BCPAP PTC −V600E High High KTC-1 PTC − wt High High 8305C PTC/ATC − V600E Low High

We also examined if both the MAPK/ERK and PI3K/Akt pathways areimportant in mediating changes in invasion potential triggered by PDGFRactivation. We used pharmacologic blockade of either the PI3K/Akt orMAPK/ERK pathways in TPC-1 cells and assessed invasion potential asshown in FIG. 9.

Disrupted invasive potential of TPC-1 cells with small moleculeinhibition of (A) PI3K/Akt (Ly294002) or (B) MAPK/ERK (U0126) pathwayswith Western blots confirming decreased protein expression is shown inFIG. 9. The invasive assay (C) confirms that blockade of either pathwayis sufficient to abrogate increases in invasive potential mediated byPDGF-BB stimulation. Cell viability is not significantly altered by thesmall molecule inhibitors and does not confound our results (D).RFU=relative fluorescence units.

Western blots of PI3K/Akt blockade (Ly294002) and MAPK/ERK blockade(U0126) in PDGF-BB stimulated TPC-1 cells are shown in FIG. 9. It isclear that blockade of either pathway prevents PDGF-BB mediatedincreases in invasive potential of the TPC-1 cell line (FIG. 9C). Forboth pathways, the blockade was nearly complete relative to controls andthe viability of the cell lines was not significantly altered. Based onthis data, and the Western blots outlined above (FIG. 8), it appearsthat both the MAPK/ERK and PI3K/Akt pathways play an important role inmediating the effects of PDGFR activation and increased invasivepotential.

Discussion

As shown herein, PDGFR-α is strongly associated with lymph nodemetastases in papillary thyroid cancer. Also, PDGFR-β does not appear tobe linked to metastatic disease despite the fact it is clearly expressedin the majority of cancer specimens we surveyed. Neither the α- orβ-subunits is expressed at significant levels in normal thyroid tissue.The association of PDGFR-α with a more aggressive, metastatic phenotypein PTC patient specimens was mirrored in studies of invasive potentialin PTC experimental cell lines. We demonstrate that PDGFR-mediatedchanges in invasive potential are directly and strongly related to thepresence of PDGFR-α in the different cell lines (FIG. 6). Cell lineswith only PDGFR-β did not demonstrate increased invasive potential withPDGFR activation, nor did cell lines with both PDGFR-α and -β that hadselective siRNA knockdown PDGFR-α (FIG. 7). While not wishing to bebound by theory, it is believed the α-subunit is important in conveyingincreased invasive potential and the presence, or absence, of PDGFR-βdoes not appear modify invasive potential in response to PDGF-BBstimulation. The differential expression of PDGFR and its associationwith disease progression has important diagnostic considerations andimplications for therapy in the choice and design of tyrosine kinaseinhibitors to treat metastatic thyroid cancer.

Treatment of metastatic PTC, that in many cases may be resistant toradioactive iodine, is problematic with significant morbidity incurredby patients through repeated surgical resections or high-doseradioactive iodine treatments. Although comprising a relatively smallproportion of thyroid cancer patients, these individuals sufferdisproportionately and radioactive iodine resistance in thyroid cancerhas prompted trials using tyrosine kinase inhibitors to address thesedifficult cases as reviewed by Gild et al.³⁹ The drugs used thus farinclude axitinib, motesanib, sorafenib, and sunitinib. The rationale forselecting these drugs in treating thyroid cancer has essentially beenempirical, relying on observations in breast and colon cancer.⁴⁰⁻⁴² Mostof these drugs are multikinase inhibitors that target the differentPDGFR and VEGFR to varying degrees and in some cases it is not clearwhich receptor subgroup is most effectively targeted. Objectiveresponses to TKI therapy in thyroid cancer vary anywhere between zeroand 55% but because of the small number of patients treated in thesetrials, the varying treatment regimes and different outcome measures, itis difficult to draw conclusions.^(39,40) Sorafenib and sunitinib appearto have favourable outcomes and acceptable side-effect profiles thatpermit ongoing use and further phase III trials.^(38,43).

As shown herein, PDGFR-α/β signaling is mediated by both the MAPK/ERKand PI3K/Akt pathways in PTC. The cell line experiments hereindemonstrate that increased invasive potential mediated by PDGF signalingrequires the activity of both pathways. The time course for activationof both pathways was very similar with PDGF-BB stimulation in TPC-1cells and disruption of either pathway, using small molecule inhibitors,was sufficient to completely abrogate any change in invasive potentialwith PDGFR activation (FIG. 9). We also show that sunitinib, throughblockade of PDGFR signaling, can down-regulate signaling through bothpathways effectively.

In summary, PDGFR-α is associated with lymph node metastases inpapillary thyroid cancer. The selective and strong expression of theα-subunit, but not PDGFR-β, in primary tumors with lymphatic metastasespermits an immunohistochemical test to aid in identifying patients withoccult metastases. PDGFR-α also appears to confer increased invasivepotential in papillary thyroid cancer cell lines with PDGF-BBstimulation. Downstream signaling is mediated through both the MAPK/ERKand PI3K/Akt pathways and disruption of either pathway can mitigate theeffects of PDGFR-activation on cell invasion potential.

Example II Patients and Methods

Patient Specimens

Ethics approval was obtained through the University of Alberta HeathResearch Ethics Board ID Pro00018758. Specimens prepared for primarycell culture or tissue banking were placed in culture media or OCT(Optimal Cutting Temperature compound), respectively, within 10 minutesof devascularisation. For the tissue array with paraffin specimens, atotal of 124 patients were selected with papillary thyroid carcinoma, 66without and 58 with lymphatic metastases. In all cases patients had atotal thyroidectomy with a level VI lymph node dissection such thathistopathology could be used to document the true node negative cases.Two pathologists separately assessed the specimens to document primarytissue diagnosis as well as the presence of lymphatic metastases innodes sectioned.

Reagents and Antibodies

The Mek inhibitor U0126 and PI3K inhibitor Ly294002 were from CellSignaling (Danvers, USA) and used at 10 and 50 uM respectively.Sunitinib malate was purchased from TORCIS Bioscience (Ellisville, USA)and used at 0.25 umol/L. STAT3 inhibitor was purchased from Santa CruzBiotechnology, (Santa Cruz, USA). Tetramethylrhodamine ethyl ester(TMRE) was purchased from Molecular Probes.

The following antibodies were used for immunoblotting and for stainingthe paraffin tissue arrays: phospho-Erk1/2(Thr 202/Tyr204) (E10: #9106),Akt (#9272), phospho-Akt (Ser473) (587F11: #4051), PDGFR-α(D1E1E:#3174), phospho-PDGFR-α/β (Tyr849)/(Tyr857) (C43E9: #3170), wereall from Cell Signaling Technology (Danvers, USA). The PDGFR-β antibodyand total Erk1 antibody (K-23: sc94) were from Santa Cruz Biotechnology,(Santa Cruz, USA).

Cell Culture

TPC-1 and BCPAP experimental cell lines were generously provided by Dr.S. Ezzat, University of Toronto, Canada. 8305C was purchased from DSMZ(Braunschweig, Germany). RET/PTC (TPC-1) and BRAF (BCPAP, 8305C)mutation status and thyroid cell origin was confirmed using Pax-8 andTTF-1 staining.³⁷ Primary cell culture and experimental cell lines weremaintained in RPMI 1640 media supplemented with 10% FBS.

Western Blot Analyses

Cells were lysed in RIPA buffer [150 mM NaCl, 100 mM Tris (pH 8.0), 1%Triton X-100, 1% deoxycholic acid, 0.1% SDS, 5 mM EDTA, and 10 mM NaF]supplemented with 1 mM sodium vanadate, 2 mM leupeptin, 2 mM aprotinin,1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM DTT, 2 mM pepstatin, and1:100 protease inhibitor cocktail set III on ice. After centrifugationat 4° C. at 18,000 rpf for 15 min, the supernatant was harvested as thetotal cellular protein extracts, aliquoted and stored at −80° C. Theprotein concentration was determined using Bio-Rad protein assay reagent(Richmond, USA). Aliquots of protein extract samples were separated bySDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred tonitrocellulose membrane. Membranes were blocked with 5% nonfat milk inTBS containing 0.05% Tween-20 for 60 min, followed by incubation withprimary antibodies 4° C. overnight. Protein bands were detected byincubation with horseradish peroxidase-conjugated antibodies (PierceBiotechnology, Rockford, Ill., USA) and visualized with SuperSignal WestPico chemiluminescence substrate (Thermo Scientific, Rockford, Ill.,USA).

Short Hairpin (shRNA) Stable Transductions

To selectively and stably silence the expression of the PDGFR-alpha and-beta receptors in the TPC-1, BCPAP and 8305C cell lines we used theHuSH-29 shRNA Vector system (HuSH-29 shRNA Retroviral Vector Systems;OriGene Technologies, Inc.). Briefly, to silence the expression of thePDGFR-alpha receptor PTC cells were transduced with the pRS shRNAretrovirus system (Puro+) followed by selection in puromycin (2.5ug/mL). Resistant cells were assessed by western blot to select thesequences that produced the highest levels of protein expressionknock-down. The sequences used for these studies wereGATGCCTGGCTAAGAATCTCCTTGGAGCT for the TPC-1 cell line and

AGTTCCACCTTCATCAAGAGAGAGGACGAfor the 8305C cell line. To selectively knock down the PDGFR-betareceptor were transduced with the pGFP-BR-S shRNA retrovirus system(BSD+) followed by selection in blasticidin (500 ug/mL). Resistant cellswere again assessed by western blot to select the sequences thatproduced the highest levels of protein expression knock-down. Thesequences selected for these studies were TGCCTCCGACGAGATCTATGAGATCATGCfor the TPC-1 cell line and

ACCTTCTCCAGCGTGCTCACACTGACCAA

Transient transfections of TCP-1, 8305C cells (3×10⁶ cells) wereperformed using the Electro square electroporator BTX ECM 800 (225V, 8.5ms, 3 pulses). 1 nmol/L of siRNA or scrambled control was used in 3million of TPC-1 and 8305C cells. The efficiency of target geneinhibition was assessed after 48 hours transfection by using Westernblotting. TPC-1, BCPAP and 8305C were transfected with either PDGFR-αsiRNA or scramble siRNA, and starved overnight prior test. siRNA forPDGFR-α and scrambled siRNA were purchased from Siegen (Foster, Calif.,USA). Transfected PTC cell lines were plated at a density of 10,000 or20,000/ml and cultured for 5 days. Invasive cells passed throughbasement membrane layer and dissociated from the membrane usingdetachment buffer and quantified using CyQuant GR fluorescent dye.

Wound Healing, Clonogenic and Transwell Invasion Assays

Cytoselect™ 24-well cell invasion basement membrane assay kit (CellBiolabs, San Diego, USA) was used to measure the invasive properties ofthe cells. Briefly, the stable TPC-1 cell lines were seeded at a densityof 3×10⁵ cells/well and cultured for 48 hours, as previouslydescribed³⁷. Invasive cells passed through the basement membrane layer,dissociated using detachment buffer and then quantified by means ofCyQuant GR fluorescent dye.

Adherent colony formation assays were performed as described (REF#1).Fifty or 100 cells per well were plated in six-well plates, fed 5% FBSsupplemented growth medium and allowed to form colonies for 20 days.Colonies were stained with 0.5% crystal violet solution in 25% methanoland counted. The methylcellulose was used assess anchorage-independentgrowth capabilities of the cell lines.

For the wound healing assay, cells were plated in 6 well plates at80-90% confluence. A wound was created by manually scratching the cellmonolayer with a p1000 pipet tip. Cellular debris was washed with PBSand the cells were fed with complete growth medium or serum-free medium.Images and measurements were acquired at times 0, 20 and 44 hours afterwound creation.

Proliferation and Apoptosis Assays

To document the effect of PDGFR silencing on proliferation, cultureswere incubated in regular or serum-free-medium and enumerated daily for5 days with an electronic cell counter (Coulter Model Zf). The MTS assay(Promega, Madison, USA) was also performed in 8-16 replicates after 48and 72 hours of growth.

Statistical Analysis

Data were expressed as the mean±S.E. from a minimum of three independentexperiments. Statistical analyses were performed with a completelyrandom design one-way ANOVA. The correlations between protein expressionand metastatic status were assessed using Fisher's exact test for tablesand Spearman rank correlation for continuous variables. Statisticaltests are two-tailed with a P value <0.05 considered to be statisticallysignificant. The SAS computer program SAS (r) 9.2 (TS 1M0) was used toperform the analysis.

Results II

The Experiments of FIG. 10 depict selective knock down of thePDGFR-alpha and -beta subunits in the TPC-1 cell lines. In Panel (a)protein expression levels were assessed by immunoreactivity to thePDGFR-alpha or PDGFR-beta antibodies. In Panel (b) activity of signalingmolecules in the STAT3, PI3K and MAPK pathways as well as expression ofPDGF-BB was documented by immunoblotting with phosphospecificantibodies. PDGFR-alpha signaling is strongly linked to PI3K/Aktpathway. In Panel (c) growth rate was assessed in serum free medium andcells enumerated at 24, 48, 72 and 96 hours. In Panel (d) proteinexpression of thyroid differentiation markers was assessed byimmunoreactivity to the TTF-1 and Pax8 antibodies. It is noted thatPDGFR-alpha expression is linked to dedifferentiated cells lackingexpression of TTF-1 but when PDGFR-alpha is knocked-down expression ofthe differentiation marker is restored. In Panel (e) tumour suppressorprotein Rb and cell cycle marker protein D1 were documented byimmunoblotting. While total levels of Rb varied the relativephosphorylated Rb to total Rb ratio did not change significantlyindicating that cell cycle was not altered by PDGFR-alpha or -betaexpression. This was confirmed in panel (f) where cells weresynchronized at the G1/S border by a double thymidine block thenreleased and followed with propidium iodide staining at the indicatedtime points and flow cytometry analysis. There was no difference betweenthe different cell lines expressing PDGFR-alpha or -beta in cell cycleanalysis.

Increased cell size and colony formation are indicative features ofcancer cells taking on a migratory and more aggressive phenotype.

The experiments of FIG. 11 depict that expression of PDGFR-alpha andknockdown of the PDGFR-beta subunit increases colony formation and cellsize. In Panel (a) TPC-1 colony formation assay demonstrates thatPDGFR-alpha, when signaling without PDGFR-beta, induces more colonies toform. In Panel (b) TPC-1 cell size also increased when PDGFR-alpha isexpressed in the absence of PDGFR-beta. In Panel (c)TPC-1-representative photographs of cells with the various knock-downsof PDGFR-alpha or -beta. In Panel (d) BCPAP—a decrease is evident inexpression of TTF-1 differentiation marker with expression ofPDGFR-alpha as assessed by immunoreactivity to the PDGFR-alpha, TTF-1,Pax8 and Akt antibodies. In Panel (e) as with TPC-1, the cell sizeincreases with PDGFR-alpha expression in BCPAP cells. In Panel (f)BCPAP-representative photographs. Scale bar, 50 μm. The experiments ofFIG. 12 depict that cancer cell lines stably expressing shRNA showsimilar invasive potential as siRNA treatment, namely that PDGFR-alphasubunit alone drives invasive potential in TPC-1 cell lines. Invasivepotential of cells where PDGFR-beta expression is knocked down, but notPDGFR-alpha is increased as shown using the basement membrane cellinvasion assay kit. After 48 hours incubation, invasive cells weredissociated, lysed, and quantified by CyQuant GR Dye.

The experiments of FIG. 13 depict knock down of PDGFR-alpha or -betasubunit results in opposing effects in tumour formation on a micexenograft model. Mice (4 or 5 animals per group as indicated) wereinoculated with sh NT (Scrambled), sh PDGFR-alpha (alpha knock down) orsh PDGFR-beta (beta knock down) TPC-1-derived cell lines in Matrigel.The average tumour volume (top panel) and tumour weight (bottom panel)was much higher for mice given cell lines expressing only PDGFR-alphaand statistical significance between groups were calculated 20 daysafter inoculation.

In the experiments of FIG. 14, Panel (a) shows immunohistochemicalpattern of PDGFR subunit expression in the mouse xenografts. ThePDGFR-alpha only tumors are more invasive and demonstrate a more diffusegrowth pattern than the more differentiated PDGFR-beta expressingtumors. Scale bar, 50 μm. In Panel (b) immunoblots show expression ofthyroid markers in the mice xenograft implantations demonstrating thattumors in mice with PDGFR-alpha are dedifferentiated thus lacking TTF-1expression. This correlates with the in vitro results.

In the experiments of FIG. 15, there is present the data from analysisof an SABiosciences PI3K/Akt mRNA array demonstrating that PDGFR-alphamRNA levels in metastatic specimens from human patients are more than 5times that in primary tumors. In these experiments RNA was isolated fromeither primary tumors (“1 tumor”) or metastatic tumors (“Mets”). The RNAwas converted to cDNA, and analyzed using an SABiosciences PCR Array(PI3K-AKT Signaling PCR Array) which contains a preset mix of primersfor genes in the signaling pathway (including PDGFα), together with thesoftware to analyze the result.

Discussion II

From the foregoing experiments it is evident that expression of PDGFRalpha leads to two main changes in cell lines; the first is that thecells become more invasive as based on invasion assays and secondly, inmultiple cell lines, PDGFR alpha leads to dedifferentiation, both ofwhich are associated with more aggressive metastatic tumors.

This was further demonstrated in the experiments in which tumors in micefrom transplanted PDGFR alpha expressing thyroid cancer cells werelarger by weight and volume than cell lines that only expressed PDGFRbeta. The combination of PDGFR alpha and beta demonstrated anintermediate phenotype compared to the slow growing, less aggressivePDGFR beta only expressing cells and the most aggressive PDGFR alphaonly cells.

PDGFR subunits alpha and beta thus modulate the others activity in cellssuch that the cell phenotype can be determined depending on whetheralpha, beta or both subunits is expressed. This is consistent with theclinical specimens described herein, where most of the thyroid cancersexpressed PDGFR beta, regardless of their metastatic status, but onlythe more aggressive metastatic specimens expressed PDGFR alpha. Tumorsthat did not express PDGFR alpha did not typically demonstrate lymphaticmetastases. It was also shown that in human specimens that PDGFR-alphagene expression levels are more than five times higher in metastasesthan in primary tumours (P=0.013) (FIG. 15).

REFERENCES

-   1. Dean D S, Gharib H. Epidemiology of thyroid nodules. Best Pract    Res Clin Endocrinol Metab. 2008; 22:901-11.-   2. Gharib H, Goellner J R. Fine-needle aspiration biopsy of the    thyroid: an appraisal. Ann Intern Med. 1993; 118:282-9.-   3. Cooper D S, Doherty G M, Haugen B R, Kloos R T, Lee S L Lee S L,    Mandel S J, Mazzaferri E L, McIver B, Pacini F, Schlumberger M,    Sherman S I, Steward D L, Tuttle R M. Revised American Thyroid    Association management guidelines for patients with thyroid nodules    and differentiated thyroid cancer. American Thyroid Association    (ATA) Guidelines Taskforce on Thyroid Nodules and Differentiated    Thyroid Cancer, Thyroid. 2009; 19:1167-1214.-   4. Crowe A, Linder A, Hameed O, et al. The impact of implementation    of the Bethesda system for reporting thyroid cytopathology on the    quality of reporting, “risk” of malignancy, surgical rate, and rate    of frozen sections requested for thyroid lesions. Cancer Cytopathol.    2011 [epub ahead of print]-   5. Yip L, Kebebew E, Milas M, Carty S E, Fahey T J 3rd, Parangi S,    Zeiger M A, Nikiforov Y E. Summary statement: utility of molecular    marker testing in thyroid cancer. Surgery. 2010; 148:1313-5.-   6. Udelsman R. Treatment of persistent or recurrent papillary    carcinoma of the thyroid—the good, the bad, and the unknown. J Clin    Endocrinol Metab. 2010; 95:2061-2063-   7. Tee Y Y, Lowe A J, Brand C A, Judson R T. Fine-needle aspiration    may miss a third of all malignancy in palpable thyroid nodules: a    comprehensive literature review. Ann Surg. 2007; 246:714-20.-   8. Shaha A R, Shah J, Loree T R. Patterns of failure in    differentiated carcinoma of the thyroid based on risk groups. Head    Neck. 1998; 20:26-30.-   9. Machens A, Hinze R, Thomusch O, Dralle H. Pattern of nodal    metastasis for primary and reoperative thyroid cancer, World J Surg.    2002; 26:22-28.-   10. Ito Y, Miyauchi A. Lateral lymph node dissection guided by    preoperative and intraoperative findings in differentiated thyroid    carcinoma. World J Surg. 2008; 32:729-39.-   11. Rotstein L. The role of lymphadenectomy in the management of    papillary carcinoma of the thyroid. J Surg Oncol. 2009; 99:186-188.-   12. Sywak M, Cornford L, Roach P, Stalberg P, Sidhu S, Delbridge L.    Routine ipsilateral level VI lymphadenectomy reduces postoperative    thyroglobulin levels in papillary thyroid cancer. Surgery. 2006;    140:1000-1005-   13. Lundgren C I, Hall P, Dickman P W, Zedenius J. Clinically    significant prognostic factors for differentiated thyroid carcinoma:    a population-based, nested case-control study. Cancer. 2006;    106:524-31.-   14. Shibru D, Chung K W, Kebebew E. Recent developments in the    clinical application of thyroid cancer biomarkers. Curr Opin Oncol.    2008; 20:13-18.-   15. Taccaliti A, Boscaro M. Genetic mutations in thyroid carcinoma.    Minerva Endocrinol. 2009; 34:11-28.-   16. Marchetti I, Lessi F, Mazzanti C M, Bertacca G, Elisei R, Coscio    G D, Pinchera A, Bevilacqua G. A morpho-molecular diagnosis of    papillary thyroid carcinoma: BRAF V600E detection as an important    tool in preoperative evaluation of fine-needle aspirates. Thyroid.    2009; 19:837-842-   17. Zatelli M C, Trasforini G, Leoni S, Frigato G, Buratto M,    Tagliati F, Rossi R, Cavazzini L, Roti E, degli Uberti E C. BRAF    V600E mutation analysis increases diagnostic accuracy for papillary    thyroid carcinoma in fine-needle aspiration biopsies. Eur J    Endocrinol. 2009; 161:467-473.-   18. DeLellis R A. Pathology and genetics of thyroid carcinoma. J    Surg Oncol. 2006; 94:662-669.-   19. Nikiforov Y E. Thyroid carcinoma: molecular pathways and    therapeutic targets. Mod Pathol. 2008; 21:S37-43.-   20. Chiu C G, Strugnell S S, Griffith O L, Jones S J, Gown A M,    Walker B, Nabi I R, Wiseman S M. Diagnostic utility of galectin-3 in    thyroid cancer. Am J Pathol. 2010; 176:2067-2081.-   21. Griffith O L, Chiu C G, Gown A M Jones S J, Wiseman S M.    Biomarker panel diagnosis of thyroid cancer: a critical review.    Expert Rev Anticancer Ther. 2008; 8:1399-1413.-   22. Griffith O L, Melck A, Jones S J, Wiseman S M. Meta-analysis and    meta-review of thyroid cancer gene expression profiling studies    identifies important diagnostic biomarkers. J Clin Oncol. 2006;    124:5043-51.-   23. Shibru D, Hwang J, Khanafshar E, Duh Q Y, Clark O H, Kebebew E.    Does the 3-gene diagnostic assay accurately distinguish benign from    malignant thyroid neoplasms? Cancer. 2008; 113:930-5.-   24. Chiu C G, Strugnell S S, Griffith O L, Jones S J, Gown A M,    Walker B, Nabi I R, Wiseman S M. Diagnostic utility of galectin-3 in    thyroid cancer. Am J Pathol. 2010; 176:2067-81.-   25. Nikiforov Y E, Ohori N P, Hodak S P, Carty S E, Lebeau S O,    Ferris R L, Yip L, Seethala R R, Tublin M E, Stang M T, Coyne C,    Johnson J T, Stewart A F, Nikiforova M N. Impact of Mutational    Testing on the Diagnosis and Management of Patients with    Cytologically Indeterminate Thyroid Nodules: A Prospective Analysis    of 1056 FNA Samples. J Clin Endocrinol Metab. 2011 [epub August 31].-   26. Lee S H, Lee J K, Jin S M Lee K C, Sohn J H, Chae S W, Kim D H.    Expression of cell-cycle regulators (cyclin D1, cyclin E, p27kip1,    p57kip2) in papillary thyroid carcinoma. Otolaryngol Head Neck Surg.    2010; 142:332-337.-   27. Liang H, Zhong Y, Luo Z, Huang Y, Lin H, Luo M, Zhan S, Xie K,    Ma Y, Li Q Q. Assessment of biomarkers for clinical diagnosis of    papillary thyroid carcinoma with distant metastasis. Int J Biol    Markers. 2010; 25:308-45.-   28. Zhu X, Sun T, Lu H, Zhou X, Lu Y, Cai X, Zhu X. Diagnostic    significance of CK19, RET, galectin-3 and HBME-1 expression for    papillary thyroid carcinoma. J Clin Pathol. 2010; 63:786-9. [Epub    2010 Jul. 19].-   29. Gu L Q, Li F Y, Zhao L, Liu Y, Chu Q, Zang X X, Liu J M, Ning G,    Zhao Y J. Association of XIAP and P2X7 receptor expression with    lymph node metastasis in papillary thyroid carcinoma. Endocrine.    2010; 38:276-82.-   30. Homsi J, Daud A I. Spectrum of activity and mechanism of action    of VEGF/PDGF inhibitors. Cancer Control. 2007; 14:285-94.-   31. Provencio M, Garcia-Campelo R, Isla D, de Castro J.    Clinical-molecular factors predicting response and survival for    tyrosine-kinase inhibitors. Clin Transl Oncol. 2009; 11:428-436.-   32. Liu J, Liao S, Huang Y, Samuel R, Shi T, Naxerova K, Huang P,    Kamoun W, Jain R K, Fukumura D, Xu L. PDGF-D improves drug delivery    and efficacy via vascular normalization, but promotes lymphatic    metastasis by activating CXCR4 in breast cancer. Clin Cancer Res.    2011; 17:3638-3648.-   33. Lei H, Velez G, Kazlauskas A. Pathological signaling via    platelet-derived growth factor receptor {alpha} involves chronic    activation of Akt and suppression of p53. Mol Cell Biol. 2011;    31:1788-1799.-   34. Cornelia H, Alsinet C, Villanueva A. Molecular pathogenesis of    hepatocellular carcinoma. Alcohol Clin Exp Res. 2011; 35:821-825.-   35. Yano Y, Uematsu N, Yashiro T. Gene expression profiling    identifies platelet-derived growth factor as a diagnostic molecular    marker for papillary thyroid carcinoma. Clin Cancer Res 2004;    10:2035-43.-   36. Bruland O, Fluge O, Akslen L A et al. Inverse correlation    between PDGFC expression and lymphocyte infiltration in human    papillary thyroid carcinomas. BMC Cancer 2009; 9:425-432.-   37. Wang Y, Ji M, Wang W, Miao Z, Hou P, Chen X, Xu F, Zhu G, Sun X,    Li Y, Condouris S, Liu D, Yan S, Pan J, Xing M. Association of the    T1799A BRAF mutation with tumor extrathyroidal invasion, higher    peripheral platelet counts, and over-expression of platelet-derived    growth factor-B in papillary thyroid cancer. Endocr Relat Cancer.    2008; 15:183-90.-   38. Sherman S I. Targeted therapies for thyroid tumors. Mod Pathol.    2011 April; 24 Suppl 2:S44-52.-   39. Gild M L, Bullock M, Robinson B G, Clifton-Bligh R. Multikinase    inhibitors: a new option for the treatment of thyroid cancer. Nat    Rev Endocrinol. [Epub 2011 Aug. 23].-   40. Romagnoli S, Moretti S, Voce P, Puxeddu E. Targeted molecular    therapies in thyroid carcinoma. Arq Bras Endocrinol Metabol. 2009;    53:1061-73.-   41. Gupta-Abramson V, Troxel A B, Nellore A et al. Phase II trial of    sorafenib in advanced thyroid cancer. J Clin Oncol. 2008; 26:4714-9.-   42. Kloos R T, Ringel M D, Knopp M V et al. Phase II trial of    sorafenib in metastatic thyroid cancer J Clin Oncol. 2009;    27:1675-1684.-   43. Can L L, Mankoff D A, Goulart B H, Eaton K D, Capell P T, Kell E    M, Bauman J E, Martins R G. Phase II study of daily sunitinib in    FDG-PET-positive, iodine-refractory differentiated thyroid cancer    and metastatic medullary carcinoma of the thyroid with functional    imaging correlation. Clin Cancer Res. 2010; 16:5260-8.-   44. Brose M S, Nutting C M, Sherman S I, Shong Y K, Smit J W, Reike    G, Chung J, Kalmus J, Kappeler C, Schlumberger M. Rationale and    design of decision: a double-blind, randomized, placebo-controlled    phase III trial evaluating the efficacy and safety of sorafenib in    patients with locally advanced or metastatic radioactive iodine    (RAI)-refractory, differentiated thyroid cancer. BMC Cancer. 2011;    11:349-356.-   45. Schweppe R, Klopper J, Korch C et al. Deoxyribonucleic Acid    Profiling Analysis of 40 Human Thyroid Cancer Cell Lines Reveals    Cross-Contamination Resulting in Cell Line Redundancy and    Misidentification. J Clin Endocrinol Metab 2008; 93:4331-4341.-   46. Wang P, Wu F, Zhang J, McMullen T, Young L C, Ingham R J, et al.    Serine phosphorylation of NPM-ALK, which is dependent on the    auto-activation of the kinase activation loop, contributes to its    oncogenic potential. Carcinogenesis. 2011; 32:146-53.-   47. Tambouret R, Geisinger K R, Powers C N, Khurana K K, Silverman J    F, Bardales R, Pitman M B. The clinical application and cost    analysis of fine-needle aspiration biopsy in the diagnosis of mass    lesions in sarcoidosis. Chest. 2000; 117:1004-1011.-   48. Morgenthau A S, Iannuzzi M C. Recent advances in sarcoidosis.    Chest. 2011 January; 139(1):174-82.-   49. Russell M R, Liu Q, Lei H, Kazlauskas A, Fatatis A. The    alpha-receptor for platelet-derived growth factor confers    bone-metastatic potential to prostate cancer cells by ligand- and    dimerization-independent mechanisms. Cancer Res. 2010; 70:4195-203.-   50. Eckert M A, Lwin T M, Chang A T, Kim J, Danis E, Ohno-Machado L,    Yang J. Twist1-induced invadopodia formation promotes tumor    metastasis. Cancer Cell. 2011; 19:372-86.

All publications, patents and patent applications mentioned in thisSpecification are indicative of the level of skill those skilled in theart to which this invention pertains and are herein incorporated byreference to the same extent as if each individual publication patent,or patent application was specifically and individually indicated to beincorporated by reference.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodification as would be obvious to one skilled in the art are intendedto be included within the scope of the following claims.

1. (canceled)
 2. A method comprising: a) obtaining a sample from asubject with thyroid cancer; b) processing said sample; c) performing ananalyte binding assay comprising contacting the processed sample with areagent to form a complex between the reagent and the biomarker presentin the sample; d) generating a result using instrumentation configuredto detect said complex, said result indicative of the amount orconcentration of said complex formed to determine the amount orconcentration of said biomarker in the sample; and e) administering atreatment for metastatic papillary thyroid cancer to said subject whenthe amount of the biomarker in the sample is greater than that in acontrol sample, wherein said biomarker is PDGFR-α.
 3. (canceled)
 4. Themethod of claim 2, wherein said biomarker is a biomarker protein, abiomarker transcript, or biomarker activity.
 5. The method of claim 2,wherein said analyte binding assay is an immunoassay.
 6. The method ofclaim 5, wherein said immunoassay is immunohistochemistry.
 7. The methodof claim 2, wherein said analyte binding assay is an RNA detectingassay.
 8. The method of claim 7, wherein said RNA detecting assaycomprises RT-PCR or in situ hybridization.
 9. The method of claim 6,wherein said immunohistochemistry is performed with an automated system,or a manual system.
 10. The method of claim 2, wherein said assayresults are quantitative or semi-quantitative.
 11. The method of claim2, wherein said processing comprises formalin fixing said sample,paraffin-embedding said sample, snap freezing said sample, treating saidsample to isolate DNA, RNA, or protein, or any combination thereof. 12.The method of claim 2, wherein said treatment comprises surgicalresection, radio therapy, chemotherapy, or combinations thereof.
 13. Themethod of claim 12, wherein said radio therapy comprises radio iodineablative therapy.
 14. The method of claim 12, wherein said chemotherapycomprises a tyrosine kinase inhibitor such as sorafenib, sunitinib,axitimb, or motisanib.
 15. The method of claim 2, wherein said treatmentcomprises administering an inhibitor of PDGFR-α to said subject.
 16. Themethod of claim 15, wherein said inhibitor comprises an RNA interferencemolecule, a small molecule, a nucleic acid, an antibody, a peptide, apharmaceutical composition, an aptamer, or combinations thereof.
 17. Themethod of claim 16, wherein said RNA interference molecule comprises aRNAi molecule, a siRNA molecule, or a shRNA molecule.
 18. A system fortreating a subject with or suspected of having metastatic papillarythyroid cancer, comprising: a) a reagent which specifically binds fordetection of PDGFR-α in a tumor sample from a patient with thyroidcancer, and b) an assay instrument configured to receive a tumor sampleand contact the reagent with the tumor sample, and to generate one ormore assay result indicative of binding said reagent with the PDGFR-αwithin the tumor sample which is assayed for specific binding, whereinsaid assay instrument comprises a detector set to detect a complexbetween said reagent and the PDGFR-α within the tumour sample, andwherein the instrument generates an assay result, and wherein thereagent is specific for PDGFR-α protein, PDGFR-α transcript, or PDGFR-αactivity. 19-20. (canceled)
 21. The system of claim 18, furthercomprising a treatment for metastatic papillary thyroid cancer for saidsubject when the amount of the PDGFR-α in the sample is greater thanthat in a control sample. 22-27. (canceled)
 28. A kit for treating asubject with or suspected of having papillary thyroid cancer,comprising: a) a reagent for performing an analyte binding assaycomprising contacting a processed sample from a subject with thyroidcancer with said reagent to form a complex between the reagent and abiomarker present in the sample, wherein said biomarker is PDGFRα; andb) instructions for treating a subject with or suspected of havingmetastatic papillary thyroid cancer according to the method of claim 2.29. The kit of claim 28, wherein said reagent comprises an agent whichbinds to PDGFRα transcript or PDGFRα protein.
 30. The kit of claim 29,wherein said reagent comprises an antibody. 31-46. (canceled)